The first step in viral infection is attachment of the virus to the cell surface and cell surface carbohydrates play an important role in this process. Due to the broad array of structural motifs possible with carbohydrates compared to proteins, many viruses and pathogens utilize carbohydrates as initial cell attachment receptors. The carbohydrate moieties mediating these interactions are modified proteins or lipids in the form of glycoproteins and glycosphingolipids, respectively, or exist as glycosaminoglycan (GAG) chains attached to proteins in the form of proteoglycans.
As a genus, the Dependoparvoviruses, belonging to the ssDNA packaging Parvoviridae, use a diverse group of cell surface carbohydrates for attachment, entry, and cellular transduction. Adeno-associated virus serotype 2 (AAV2), AAV3B, AAV6, and AAV13 bind to heparin sulfate proteoglycans (HSPGs) (1-7). However, these viruses differ in their affinity and specificity for HS (1). AAV1, AAV4, AAV5, and AAV6 all use different forms of sialic acid (SIA) (7-10). While both AAV4 and AAV5 require the α2-3 form of SIA, treatment of cells with specific glycosylation inhibitors and re-sialation experiments with neuraminidase-treated erythrocytes demonstrated that AAV4 preferentially attached to an α2-3 SIA present on an O-linked carbohydrate core, and AAV5 attached to the N-linked type (8). Analysis of AAV1 and AAV6 determined that both use either α2,3-linked or α2,6-linked SIA when transducing numerous cell types and that SIA supersedes HS in controlling AAV6 transduction (7, 10). Similarly, an AAV isolate found as a contaminate in a stock of bovine adenovirus termed BAAV also requires cell surface SIA for transduction and internalization but the terminal SIA groups must be linked to a glycoshingolipid core of a ganglioside (11).
Carbohydrate structural motifs are not static and their presentation on the cells' surface varies with cell differentiation and maturation, all of which can affect viral attachment. Furthermore, their polarized surface expression or presence in the extracellular matrix or fluids, such as saliva or bronchoalveolar lavage fluid, can affect and block a virus's attachment to a cell. For example, the protective mucins secreted by airway epithelia are heavily glycosylated with an abundance of O-linked SIA. Binding and competition experiments demonstrate that AAV4 will bind to and is inhibited by purified muc-1 but not its deglycosylated form (9). In contrast, AAV5 only weakly binds muc-1 and its transduction is not inhibited in competition experiments or by bronchoalveolar fluid (9, 12).
Extensive mutagenesis on AAV2 localized its HS binding region to a basic patch of amino acids, with R585 and R588 (AAV2 VP1 numbering), located close to the top of the protrusions that surround the icosahedral threefold symmetry axis of the capsid, shown to be the most critical for this interaction (13, 14). Interestingly, while a mutation in this region blocks virus binding and transduction in vitro, it appears to alter but not ablate transduction in vivo. Kern et al. reported that a mutation of amino acids R585 and R588 in AAV2 results in a vector with improved specificity for heart tissue compared to WT virus, which can direct gene transfer to both the heart and liver. However, while insertion of the peptide comprising AAV2's residues 585-RGNR-588 onto AAV5 can confer heparin binding activity to this serotype; it does not confer sensitivity to heparin competition during transduction suggesting that cellular transduction by AAV5 is likely controlled by its initial SIA interaction which is not ablated by the peptide insert (14).
Adeno-associated viruses have emerged as one of the most promising vectors in the field of gene therapy. However, many current AAV vectors remain inefficient at delivering heterologous nucleic acid molecules to their intended target cells. Moreover, the ability of the immune response to recognize and mount an immune response against current AAV vectors limits the use of such vectors, particularly in cases where vectors are to be administered multiple times. Thus, a need exists for improved vectors for use in gene therapy and immunization applications. The present invention addresses such need and provides other benefits as well.